Comparative Proteomic Analysis Of Overexpressing CIPK14 And Double Knockout WHIRLY1/3 Mutation Resulted The Pale Green Leaf Of Arabidopsis

Leaf senescence is the last stage of plant growth and development,the leaf aging is induced by factors internal and environmental factors.The WHIRLY(WHY)transcription factor family is found in plants and is able to bind to single-stranded DNA.There are three family members in Arabidopsis thaliana,AtWHY1 and AtWHY3 are the type I,while AtWHY2 is type ?.WHY1 has been proved to be located in chloroplasts and nucleus and may play a key role in restrograde signal transductions and regulate the process of leaf senescence,but the detail mechanism remains unknow.Until now,most of functional researches of WHY 1 are reported on the transcriptional level.Based on our previous report that the dual localization and distribution of WHY1 protein between plastids and the nucleus were affected by WHY1 protein phorsphorylation status mediated by CIPK14,this study focuses on comparable analysis of the phenotype and proteomic alteration between double knockout WHY1/WHY3(ko1/3)and overexpressing CIPK14(oeCIPK14)lines.The total proteins of WT,kowhyl,why1why3 and oeCIPK14 rosette leaves were seperated by 2-DE to acquire significantly differential expression proteins.The selected proteins were identified in transcriptional level by qRT-PCR and in protein level by western blot.The chlorophyll content,chlorophyll fluorescence kinetic curve and rapid light responding curve were used to determine for photosynthetic performances analysis of the detail phenotype of different mutants.The main results as follows:1.Total proteins of WT,why1,why1why3 and oeCIPK14 variegated line(oeCIPK14-var)were separated by 2-DE gels,more than 800 protein spots were detected reproducibly by ImageMasterTM 2D Platinum 7.0 software.While the protein expression pattern of why1 mutant and why1why3 normal line showed no significant difference compared to WT,33 proteins were shown significantly differential expression in why1why3 variegated line(why1why3-var),34 proteins were shown significantly differential expression in oeCIPK14-var.Five overlapping proteins were detected in why1why3-var and oeCIPK14-var lines.66 proteins were successfully identified by MALDI-TOF/TOF-MS.The five overlapping proteins were ATP-dependent Clp protease proteolytic subunit-related protein(ClpR3),Ribulose bisphosphate carboxylase large chain,Beta-amylase,Ribosome-recycling factor(RRF),Ribulose bisphosphate carboxylase small chain.The results of candidate protein annotation showed that most of proteins were involved in photosynthesis,amino acids and protein metabolism and defense and antioxidation.Among them,most of the proteins in photosynthesis and defense and antioxidation were up-regulated,the proteins of calvin cycle were down-regulated in both why1why3-var and oeCIPK14-var lines.2.Firstly,26 protein encoded genes with significant differential expression were analyzed in transcriptional level by qRT-PCR using why1why3-var and oeWHY1 compared to WT.The expression of 13 genes was consistent with protein expression pattern,and 6 genes were significantly different.The expression of 14 genes was opposite to protein expression pattern,and 8 genes were significantly different.Secondly,26 genes were investigates in kocipk14 and oeCIPK14-var compared to WT,The expression of 19 genes was consistent with protein expression pattern,and 17 genes were significantly different.The expression of 8 genes was opposite to protein expression pattern,and 5 genes were significantly different.For the five overlapping protein encoded genes,the levels of ClpR3 in oeCIPK14-var and RRF in why1why3-var were consistent with protein expression level.3.Compared with WT,the content of chlorophyll of oeCIPK14-var is lower.Chlorophyll fluorescence parameters Fv/Fm,Y(?),qP and ETR decreased in ko1/3-var and oeCIPK14-var,but Y(NO)and NPQ were increased,the two variegated lines showed sensitivity for strong light response.4.The immunodetection analysis showed that psbR and psbP were down-regulated expression in oeCIPK14-var,Lhcal was up-regulated expression in why1why3-var,cytf was up-regulated expression in ko1/3-var and oeCIPK14-var.The result of psbR was consistant with 2-DE.Comprehensive analysis of above results,we concluded that the light reaction related proteins,including HCF101,ChlI-2,CP26,PPD4,TROL,cyt f were up-regulated expression in ko1/3-var and oeCIPK14-var for maintaining of the redox state of the photosynthetic electron transport chain.Several proteins in Calcin cycle,such as RuBisCO,RCA,PGK,CA were down-regulated expressin in why1why3-var and oeCIPK14-var,indecating that the accumulation of CO2 and photosynthesis efficiency decreased in ko1/3-var and oeCIPK14-var.It speculates that the excessive light energy may trigger the the accumulation of ROS in chloroplast,many defense and antioxidation related proteins were up-regulated for ROS scavenging.Our study revealed that the WHY1 may act as signaling molecule and transmit the redox state of the photosynthetic electron transport chain to nucleus,changed the expression of nuclear genes for the developmental regulation of leaf at early stage in protein level.